Sex Applicability

Life Stage Applicability

Key Event Relationship Description

The ability of a neuron to communicate is based on neural network formation that relies on functional synapse establishment (Colón-Ramos, 2009). The main roles of synapses are the regulation of intercellular communication in the nervous system, and the information flow within neural networks. The connectivity and functionality of neural networks depends on where and when synapses are formed. Therefore, the decreased synapse formation during the process of synaptogenesis is critical and leads to decrease of neural network formation and function in the adult brain.

Synaptic transmission and plasticity require the integrity of the anatomical substrate. The connectivity of axons emanating from one set of cells to post-synaptic side of synapse on the dendrites of the receiving cells must be intact for effective communication between neurons. Changes in the placement of cells within the network due to delays in neuronal migration, the absence of a full formation of dendritic arbors and spine upon which synaptic contacts are made, and the lagging of transmission of electrical impulses due to insufficient myelination will individually and cumulatively impair synaptic function. These anatomical alterations are responsible for many structural anomalies reported in various regions of the brain following severe developmental hypothyroidism. Although the primary evidence of synaptic transmission impairments in hypothyroid models have come from studying the hippocampus, it is assumed that the role thyroid hormones play in these processes is likely similar across different brain regions. Altered hippocampal structure induced by decreased TH levels impacts neurogenesis in the developing hippocampus or cortex, contributing to deficits in synaptic function.

Evidence Supporting this KER

The weight of evidence supporting the relationship between decreased synaptogenesis induced by TH insufficiency and altered neuronal network and synaptic function is moderate. Functional change as exemplified by alterations in synaptic transmission may be more easily detected than structural abnormalities. The exact alignment between the neuroanatomical effects (such as decreased synaptogenesis and alteration of GABAergic interneurons) that have been associated with developmental hypothyroidism (e.g., elicited by exposing rat dams to TPO inhibitors) and the neurophysiological impairments has not been entirely elucidated.

Biological Plausibility

Neuronal network formation and function are established via the process of synaptogenesis. The developmental period of synaptogenesis is critical for the formation of the basic circuitry of the nervous system, although neurons are able to form new synapses throughout life (Rodier, 1995). The brain electrical activity dependence on synapse formation is critical for proper neuronal communication.

Alterations in synaptic connectivity lead to refinement of neuronal networks during development (Cline and Haas, 2008). Indeed, knockdown of PSD-95 arrests the functional and morphological development of glutamatergic synapses (Ehrlich et al., 2007).

The biological plausibility of the known effects of TH insufficiency on brain structure having an impact on synaptic function and plasticity in brain is strong. Reductions in myelination of axons, cell number, dendritic arborization, and synaptogenesis have been described in models of severe hormone deprivation, as comprehensively summarized by Thompson and Potter, 2000. Because synaptic transmission relies on the integrity of synaptic contacts and the electrical and chemical transmission between pre- and post-synaptic neurons, it is well accepted that interference with process of synapse formation (morphological unit of neuronal network) will very much impact the neural network function.

Empirical Evidence

Most of the information on developmental hypothyroidism and altered synaptic function has been derived from studies of the hippocampus. It is presumed that structural changes of synaptic connectivity at certain level may lead to functional deficits in synaptic transmission and plasticity impairments (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Dong et al., 2005, Sui et al., 2005), but the precise structural aberration is not known. Within the hippocampus, area CA1 has been investigated primarily with in vitro techniques, using slices of hippocampus from animals exposed to TPO inhibitors (MMI or PTU) and measuring synaptic function across CA1-pyramidal cell synapses (Vara et al., 2002, Sui and Gilbert, 2003, Gilbert, 2004, Taylor et al., 2008). Pyramidal neurons of hypothyroid animals have fewer synapses and an impoverished dendritic arbor (Rami et al., 1986a, Madeira et al., 1992), with reductions ranging between 14.2 and 22.5% as observed in 30 and 180-day-old hypothyroid rats (as described by Madeira et al., 1992).

The other major region in hippocampus investigated in hypothyroid models is the perforant path-dentate gyrus synapse (Gilbert, 2011). Granule cells are the principal cell type of the dentate gyrus region of the hippocampal formation and receive input from cortical neurons in the entorhinal cortex. TPO inhibitors like PTU and MMI decrease the volume of the granule cell layer, the density of cells within the layer, and estimates of total granule cell number (Madeira et al., 1991). Migration of granule cells from the proliferative zone to the granule cell layer is retarded by thyroid deficiency as is dendritic arborization and synaptogenesis assessed by immunohistochemistry for the synaptic protein, synaptophysin (Rami et al., 1986b, Rami and Rabie, 1990, Dong et al., 2005). Impairments in synaptic function from both rodent and human studies are summarized below.

- Vara et al., 2002 This study assessed effects of TH on short-term synaptic plasticity (associated with short-term memory) in control vs hypothyroid rats. Specifically, dams were treated with 0.02% methylmercaptoimidazole (MMI, TPO inhibitor) continually from GD9. A group of pups were also treated with T3 (i.p. daily injections of T3 (20 μg /100 g body weight)) starting 72 h before killing. Data showed that hypothyroid rats presented increase in the Ca(2+)-dependent neurotransmitter release, indicative of altered neuronal network function (i.e., decreased paired-pulse facilitation), and these alterations were reverted by T3 administration. These synaptic changes were determined by an increase of synapsin I and synaptotagmin I levels in the hypothyroid rats, suggesting that TH modulate neurotransmitter release. These results are in contrast with those of Di Liegro et al. (1995), who showed that in primary cultures T3 induces the expression of synapsin I. This difference could be the result of the multiple differences between the two models: different brain regions (hippocampus vs. cortex), the age of the neurons (neonatal vs. embryonic), the preparations (slice vs. culture).

- Sui and Gilbert, 2003 Here developing rats were exposed in utero and postnatally to 0, 3, or 10 ppm propylthiouracil (PTU, TPO inhibitor), administered in the drinking water of dams from GD6 until PND30. Excitatory postsynaptic potentials and population spikes (indicative of neuronal network function) were recorded in area CA1 of hippocampal slices from offspring between PND21 and PND30. PTU caused at PND30 a decrease of maternal total T4 (43.9% with 3-ppm, and 65.0% with 10-ppm) compared to controls, with no change in total T3. Maternal TSH was increased above control levels in a dose-dependent manner. In pups, total T4 was depressed (by 75%) in both treated groups relative to the controls pups, and total T3 was depressed (35.8% in the 3-ppm group and 66.5% in the 10-ppm group) relative to the controls. TH insufficiency was dose-dependently associated with a reduction of paired-pulse facilitation and long-term potentiation of the excitatory postsynaptic potential and elimination of paired-pulse depression of the population spike. Excitatory synaptic transmission was increased by developmental exposure to PTU. This suggests that TH insufficiency compromises synaptic communication in area CA1 of developing rat hippocampus (involved in learning and memory).

- Gilbert, 2004 Here developing rats were transiently exposed to PTU (0 or 15 ppm), through the drinking water of pregnant dams beginning on GD18 until PND21. This regimen markedly reduced circulating levels of TH in pups (T3: ∼50% lower than control at PND21; T4: T4 ∼40% below control levels). Analysis of field potentials in area CA1 of hippocampal slices derived from adult male offspring exposed to PTU showed a reduction of somatic population spike amplitudes, with no differences in excitatory postsynaptic potentials (EPSP). Short-term plasticity of the EPSP (as indexed by paired pulse facilitation) was markedly decreased by PTU exposure. These data confirm associations between decrease of synaptic function in the hippocampus and decrease of neuronal network function as a consequence of TH insufficiency.

- Dong et al., 2005 Through gestation and lactation, iodine-deficient or hypothyroid dam rats were administered with either iodine-deficient diet or MMI (TPO inhibitor) added to drinking water. Exposure was terminated on PND30. Both treated groups showed lower concentrations of serum FT3 (~60% decrease on PND30) and FT4 (~80% decrease on PND30), smaller population spike amplitude (~50% decrease) and field-excitatory postsynaptic potential (f-EPSP, 50-60% decrease) slope induced by high-frequency stimulation (HFS). Also, TH insufficiency decreased the levels of c-fos (by ~60% at PND30 in MMI group) and c-jun proteins (by ~20% at PND30 in MMI group) in the hippocampus. C-fos and c-jun expression is regulated by synaptic activity, both play an important role in the neuroplastic mechanisms (synaptogenesis) critical to memory consolidation, and play an essential role in neuronal differentiation (Herdegen et al., 1997).

KEs proceeding the AO (learning and memory deficits), such as "Decreased synaptogenesis" (KEup) and "Decreased Neural Network Function" (KEdown) are also common to the AOP 13, entitled "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" (https://aopwiki.org/aops/13). In this AOP 13, data on lead (Pb) exposure as reference chemical are reported. While these studies do not refer to TH disruption, they provide empirical support for the same KER (Decreased synaptogenesis leads to decreased neuronal network function in developing brain) described in the present AOP.

- Otto and Reiter, 1984: At low Pb2+ levels (less than 30 µg/dl), slow surface-positive cortical potentials have been observed in children under five years old, which may reflect axodendritic inhibitory processes, whilst negative slow potentials are observed in children over five years. This is due to the fact that during maturation of the cortex, the locus of inhibitory activity shifts deeper to axosomatic connections. However, age-related polarity reversal has been observed in children with higher Pb2+ levels.

- Kumar and Desiraju, 1992: In experiments carried out in Wistar rats that have been fed with lead acetate (400 µg/g body weight/day) from PND 2 until PND 60, EEG findings show statistically significant reduction in the delta, theta, alpha and beta band of EEG spectral power in motor cortex and hippocampus with the exception of the delta and beta bands power of motor cortex in wakeful state.

- McCarren and Eccles, 1983: Male Sprague-Dawley rats have been exposed to Pb2+ from parturition to weaning though their dams' milk (dams received drinking water containing 1.0, 2.5, or 5.0 mg/ml lead acetate). Starting from 15 weeks of age, the characteristics of the electrically elicited hippocampal after discharge (AD) and its alteration by phenytoin (PHT) showed significant increase in primary AD duration only in the animals exposed to the higher dose of Pb2+, whereas all groups responded to PHT with increases in primary AD duration.

The exact mechanism by which a change in cell number, reduced dendritric arborization and synaptogenesis may lead to decreased neuronal network function has not been fully elucidated. Dose-dependent reductions in synaptic function in hippocampus have been demonstrated in models of moderate degrees of TH reduction, but studies of the anatomical integrity of the specific cell populations examined electrophysiologically have largely been evaluated in models of severe hypothyroidism and often in brain regions distinct from the hippocampus.

Uncertainties and Inconsistencies

The exact mechanism by which a change in cell number, reduced dendritric arborization and synaptogenesis may lead to decreased neuronal network function has not been fully elucidated. Dose-dependent reductions in synaptic function in hippocampus have been demonstrated in models of moderate degrees of TH reduction, but studies of the anatomical integrity of the specific cell populations examined electrophysiologically have largely been evaluated in models of severe hypothyroidism and often in brain regions distinct from the hippocampus.

Quantitative Understanding of the Linkage

There are no data on the quantitative linkages between altered (hippocampal or cortical) synaptogenesis and impaired neuronal network function. Developmental window of exposure and duration of exposure can modulate response-response relationships.

Response-response Relationship

Time-scale

Known modulating factors

Known Feedforward/Feedback loops influencing this KER

Domain of Applicability

The main proof of evidence comes from in vivo studies in rodents. However, Colón-Ramos (2009) has recently reviewed the early developmental events that take place during the process of synaptogenesis in invertebrates, pointing out the importance of this process in neural network formation and function. The experimental findings reviewed in this paper derive from knowledge acquired in the field of neuroscience using C. elegans and Drosophila; at the same time, emerging findings derived from vertebrates are also discussed (Colón-Ramos, 2009).